With the advent of molecular cloning techniques, an expanding array of genes with mutations responsible for important human diseases have been identified and isolated. Absent or mutated genes in human patients can be replaced by ex vivo techniques, which include transformation of cells in vitro with naked DNA, DNA encapsulated in liposomes, appropriate integration vectors followed by introduction into a host organ (“ex vivo” gene therapy).
Gene therapy provides a means for transfer of a desired gene into a subject with the subsequent in vivo expression thereof. Gene transfer can be accomplished by transfecting the subject's cells or tissues ex vivo and reintroducing the transformed material into the host. Alternatively, genes can be administered directly to the recipient.
Nabel et al., Science (1990) 249: 1285-1288, pertains to in vivo intra-arterial transfection of pigs with liposomes containing a β-gal expression plasmid. Site-specific gene expression was observed in the arterial wall. There are several drawbacks to ex vivo therapy. For example, if only differentiated, replicating cells are infected, the newly introduced gene function will be lost as those cells mature and die. Ex vivo approaches also can be used to transfect only a limited number of cells and cannot be used to transfect cells which are not first removed from the body.
As described above, in gene therapy, it is very important to appropriately select a gene to be introduced, target cells in which the introduced gene is to be expressed, gene transfer methods suitable for target tissues, and the administration route.
Cystic fibrosis (CF) is an autosomal recessive genetic disease causing inborn error of metabolism. CF patients are frequently found in the U.S. and Europe, and one in every 2,000 to 2,500 infants suffers from this disease. As a major symptom, abnormal external secretion produces viscous secreta, which are accumulated in organs such as lung, respiratory tracts, pancreas, liver, and small intestine. The current therapy of CF focuses on lung transplantation and antibiotic treatment of pulmonary infectious diseases, which is particularly fatal.
The causative gene of CF, cystic fibrosis transmembrane conductance regulator (CFTR) gene, has been identified (Riordan, J. R. et al., Science 245: 1066-1073, 1989), and it is expected to develop gene therapy for CF in which a vector carrying a normal CFTR gene is introduced to airway epithelia. In gene therapy for CF, the exogenous gene should be introduced in vivo because ex vivo treatment cannot be applied to lung and upper airway.
Several attempts have been made to administer vectors to lung. Hazinski et al. (Am. J. Respir. Cell Mol. Biol. (1991) 4: 206-209) discloses liposome-mediated gene transfer of DNA into the intact rodent lung. Cationic liposomes were complexed to three fusion gene constructs composed of 1) the chloramphenicol acetyltransferase (CAT) gene linked to a Rous sarcoma virus (RSV) promoter; 2) the CAT gene linked to a mouse mammary tumor virus (MMTV) promoter; and 3) a cytomegalovirus-β-galactosidase (CMV-β-gal) fusion gene. The liposome/DNA complexes were instilled into the cervical trachea of rats and detectable levels of gene expression observed.
Brigham et al. (Am. J. Med. Sci. (1989) 298: 278-281) describes the in vivo transfection of murine lungs with the CAT gene using a liposome vehicle. Transfection was accomplished by intravenous, intratracheal or intraperitoneal injection. Both intravenous and intratracheal administration resulted in the expression of the CAT gene in the lungs. However, intraperitoneal administration did not.
Canonico et al. (Clin. Res. (1991) 39: 219A) describes the expression of the human α-1 antitrypsin gene, driven by the CMV promoter, in cultured bovine lung epithelial cells. The gene was added to cells in culture using cationic liposomes. The experimenters also detected the presence of α-1 antitrypsin in histological sections of the lung of New Zealand white rabbits following the intravenous delivery of gene constructs complexed to liposomes.
Furthermore, U.S. Pat. No. 5,958,893 discloses a method for introducing a gene encoding truncated CFTR using currently available vectors such as adenovirus vectors or cationic liposomes.
It was demonstrated, however, that adenovirus-mediated gene transfer to airway epithelia produced low gene transfer efficiency; low rate of uptake of adenoviral particles to the apical plasma membrane could be a cause of inefficient gene transfer, and lack of both the αβγ3 integrins and the CAR receptors which are the receptors for adenovirus, in apical surface of airway epithelial cells (Goldman, M. et al., Gene Ther. 3:811-818, 1996, Boucher, R. C., J. Clin. Invest 103: 441-445, 1999). In the case of cationic liposomes, mucus reportedly prevented their uptake, and gene transfer efficiency was improved by removal of the mucus (Kitson, C. et al., Gene Ther. 6: 534-546, 1999, Zabner, J. et al., J. Biol. Chem. 270: 18997-19007, 1995, Fasbender, A. et al., Gene Ther. 4: 1173-1180, 1997).
To date, no report is available for vector systems and gene transfer methods enabling efficient introduction of exogenous genes to airway epithelia. It has thus been desired to develop vectors for efficient gene transfer to airway epithelia.
Sendai virus belonging to the family Paramyxoviridae is very useful as a vector for gene transfer, and its development is in progress (Kato, A. et al., EMBO J. 16: 578-598, 1997, WO97/16538, WO97/16539). Sendai virus shows low toxicity and expresses genes introduced therein at an extremely high level. This virus is also very safe because a gene insert in the virus vector is never integrated into the host chromosome. It has been reported that transfectionability of a Sendai virus vector is different from that of adenovirus (Goldman, M. et al., Gene Ther. 3: 811-818, 1996, Boucher, R. C., J. Clin. Invest 103: 441-445, 1999). For example, adenovirus is likely to infect injured sites, compared to uninjured sites (Kitson, C. et al., Gene Ther. 6: 534-546, 1999, Zabner, J. et al., J. Biol. Chem. 270: 18997-19007, 1995, Fasbender, A. et al., Gene Ther. 4: 1173-1180, 1997). These reports suggest that Sendai virus can complement the defect of adenovirus.